Supplementary Figure 1 YAP negatively regulates IFN- signaling. (a) Immunoblot analysis of Yap knockdown efficiency with sh-yap (#1 to #4 independent constructs) in Raw264.7 cells. (b) IFN- -Luc and PRDs I-III-Luc activity in Raw264.7 cells depleted for Yap and stimulated with SeV for 12 h. Results are shown as mean + sd of triplicates of at least two independent experiments. *P < 0.05 (two-tailed Student's t-test.) (c) qpcr analysis of sh-yap #1 efficiency (left panel) and IFNB1 mrna level (right panel) in control and YAP depleted THP1 cells followed by SeV infection at the indicated time points. (d) IFN- -Luc and PRDs I-III-Luc activity in HEK293T cells transfected with respective reporters and YAP2 or YAP4 expression plasmids followed by stimulation with SeV for 12 h. (e) qpcr analysis of HEK293T cells transfected with empty vector (Co.vec) and YAP2 or YAP4 expression plasmids for 48 h followed by Sev infection (left) or Poly (I:C) transfection at the indicated time points. All qpcr results are shown as mean + sd of triplicates of at least two independent experiments. *P < 0.05 and **P < 0.01 (two-tailed Student's t-test.) (f) Fluorescence microscopy of VSV GFP levels in HEK293T cells transfected with YAP2 or YAP4 expression plasmids followed by infection for 12 h with VSV GFP (MOI 0.1) (bright-field, upper; fluorescence, bottom). Scale bars, 100 m. Representative results are shown of least two independent experiments.
Supplementary Figure 2 YAP deficiency potentiates IFN- signaling. (a, b) qpcr analysis of Ifnb1, Ccl5 and Cxcl10 mrna levels in wild type and Yap1 +/- mouse bone marrow derived macrophages (BMDMs) infected with SeV (a) or stimulated with 5 -ppprna for the indicated time points. (c, d) qpcr analysis of Ifnb1 levels in wild type and Yap1 +/- BMDMs transfected with Poly (I:C) (c) or infected with HSV-1 (MOI, 10) (d) for the indicated time points.(e, f) qpcr analysis of Ifnb1, ccl5 and Cxcl10 mrna levels in wild type and Yap1 +/- MEFs infected with SeV (e) or stimulated with 5'-ppp RNA (f) for the indicated time points. (g, h) qpcr analysis of Ifnb1 levels in wild type and Yap1 +/- MEFs transfected with Poly
(I:C) (g) or infected with HSV-1 (MOI, 10) (h) for the indicated time points. All qpcr results are shown as mean + sd of triplicates of at least two independent experiments. *P < 0.05 and **P < 0.01 (two-tailed Student's t-test.) (i) Survival of Yap1 +/+ and Yap1 +/ mice (n=10 for each group) infected intraperitoneally with VSV (5 10 8 PFU per mouse). *P < 0.05 ( two-way analysis of variance (ANOVA)). (j) Representative hematoxylin-and-eosin-stained images of lung sections from mice as in a. Scale bars, 100 m. (k) HSV-1 viral titers in brain of Yap WT and heterogeneous mice (n = 6 mice per group) infected with HSV-1 for 72 h. (l) Survival of Yap1 +/- and WT mice (n = 10 mice per group) after intravenous injection of HSV-1 (2 10 7 PFU per mouse). *P < 0.05, ***P < 0.01 (two-tailed Student's t-test in k or two-way analysis of variance (ANOVA) in l).
Supplementary Figure 3 IFN- signaling is upregulated in YAP-deficient macrophages. qpcr of Ifnb1, Cxcl10 and Ccl5 mrna levels in Yap1 fl/fl Lyz2-Cre + and Yap1 fl/fl Lyz2-Cre - peritoneal macrophages infected or stimulated with SeV, 5'-pppRNA, VSV(MOI=1) or HSV-1 (MOI=0.1). All qpcr results are shown as mean + sd of triplicates of at least two independent experiments. *P < 0.05 and **P < 0.01 (two-tailed Student's t-test.)
Supplementary Figure 4 YAP retains IRF3 in the cytoplasm. (a) IFN- -Luc activity (left) and IFNB1 mrna in HEK293T cells depleted with YAP and transfected with cgas+sting, RIG-I N-terminal 2CARD (RIG-IN), MAVS, TBK1, IKK or IRF3-5D expression plasmids as indicated. Results are shown as mean + sd of triplicates of at least two independent experiments. *P < 0.05 (two-tailed Student's t-test.) (b) Immunoblot (IB) of total cell lysate (TCL) and immunoprecipitate derived from HEK293T cells transfected with Myc-IRF3 and Flag-YAP1 or 2 expression plasmids as indicated. (c) IB of IRF3 dimerization (Native gel), p-irf3, p-tbk1, p-ikk, total IRF3, TBK1 or IKK in HEK293T cells transfected with Flag-YAP2 expression plasmid and treated with SeV for the indicated time points. (d) IB of nuclear and cytoplasm fractions derived from HEK293T cells transfected with IRF3-Flag, IRF3-5D-Flag and
YAP4-HA as indicated. (e) IB of TCL and immunoprecipitates derived from HEK293T cells transfected with Flag-Importin 5 or Flag-Importin 1 along with IRF3-5D-Myc. Data are representative of three independent experiments with similar results (b-f).
Supplementary Figure 5 YAP is promoted for lysosomal degradation by IKK. (a) Immunoblot of immunoprecipitates derived from HEK293T cells transfected with Flag-YAP2 plasmid and treated with SeV or poly (I:C) for 12 h. Cells were treated with NH 4 Cl (10 mm) for 4 h before harvest. (b) IB analysis for endogenous YAPs of control or IKK depleted MCF10A cells treated with SeV for 12 h. (c) IB of HEK293T cells transfected with Flag-YAP4 and Myc-IKK and treated with NH4Cl (10 mm), Chlq (100 M), MG132 (10 M) and control DMSO for 6 h. Data are representative of three independent experiments with similar results (a-c).
Supplementary Figure 6 YAP is phosphorylated by IKK. (a) Immunoblot of HEK293T cells transfected with expression vectors for Flag-YAP2 wt or Flag-YAP2 5SA (in which all the previously reported by Lats1 or 2 and CK1 phosphorylated Serines at positions 61, 109, 127, 128, 131, 163, 164, and 381 are changed to Alanine), with or without Myc-IKK expression vectors as indicated. (b) Mass spectrometry identification of 4 phosphorylation sites in Flag-YAP2 5SA targeted by IKK.
(c) IB of HEK293T cells transfected with Myc-IKK and Flag-YAP2 5SA derivatives carrying the indicated additional phosphorylation site mutations. (d) Sequence alignment of IKK -mediated phosphorylation sites in YAP orthologues of different species. (e) IB of cell lysates of HEK293T cells transfected with Flag-YAP4 WT, 4SA or S403A mutant with or without Myc-IKK expression plasmid as indicated. (f) Validation of the antibody against phosphor-serine 403 of YAP. Parental, YAP1-deleted (KO) and YAP S403A-mutated (KI) HEK293T cells were treated with SeV for 8 h. Chlq (100 M) were added to the cells 6 h before harvest. Cells were then harvested for immunoprecipitation with anti-yap antibody followed by anti-p-s403 YAP immunoblot analysis. Data are representative of three independent experiments with similar results (a,c,e,f).
Supplementary Figure 7 IKK -mediated phosphorylation of YAP at Ser403 is critical for the innate antiviral response. (a) Immunoblot of IRF3 dimeration, and p-irf3, p-tbk1, p-ikk, total IRF3, TBK1 and IKK in parental and YAP S403A knock-in (KI) A549 cells. Data are representative of three independent experiments with similar results. (b) qpcr analysis of IFNB1 mrna levels (left panel) and ELISA analysis of IFN- secretion in parental and YAP S403A-mutated A549 cells stimulated with SeV, VSV (MOI, 0.1) or transfected with cgas and STING or poly (da:dt) (right panel) for 12 h. qpcr and ELISA results are shown as mean ± sd of triplicates of at least two independent experiments.